Microfluidic systems and combined microfluidic-microfluidic systems are attractive for diagnostics and allow for resource-limited settings because they use entire analytic protocols including sample pre-treatment, sample/reagent manipulation, separation, reaction, and detection integrated into a single platform. Current methods for lysing cells are based on mechanical lysis, thermal lysis, chemical lysis or electrical lysis. Once cells or samples have been lysed, or the nucleic acid is freed from the sample, microfluidic sensing systems require the nucleic acid to be purified or concentrated before delivery to a sensor. A wide range of nucleic acid extraction methods is available, each applying different types of chemistry and optimized for particular sample types. Because of their complex nature, most of the existing extraction methods are not appropriate for incorporation in microfluidic platforms or result in a significant loss of nucleic acids during the extraction step.
A variety of biological samples are taken from individuals to evaluate diagnostic and prognostic indicators of disease. Fresh tissue specimens, fixed and embedded samples and fine needle aspirate biopsies (FNA) are a valuable source of material for obtaining both molecular as well as clinical information since they often come from human specimens collected for examination of the histology of biopsies for the detection of disease. Tissue that is treated with a fixative, which prepares the sample for a variety of (immune-) histochemistry procedures, undergoes a variety of cross-linking modifications between nucleic acids and amino acids (Chaw Y. F. M. et al. Biochemistry 1980, 19: 5525-5531; Metz B. et al. J. Biol. Chem 2004. 279: 6235-6243). The fixed tissue is then encased in a block of embedding material (such as agar, gelatine or wax) which is hardened and cut into slices as little as 1-2 cell layers thick for histological studies. Compared to nucleic acid extraction from other sample sources, nucleic acid extraction from fixed and embedded sample slices requires the additional step of removal of the embedding material.
The use of formalin fixation and paraffin embedding (FFPE) to fix and preserve tissue samples is almost universal. A number of conventional protocols that solubilize paraffin and liberate nucleic acids from FFPE samples are available (Gilbert M. T. P. et al., PLoS One 2007, 2(6):e537). The traditional deparaffinisation methods start with a liquefaction step which uses an organic solvent, usually xylene, followed by a nucleic acid extraction step (Goezl et al., Biochemical and Biophysical Research Communication 1985, Vol. 130 No. 1, p 118-126). Xylene has the major disadvantages of being flammable, volatile, toxic and incompatible with plastic, making it less suitable for use in automated systems.
Nucleic acid preparation from tissue slice samples typically requires a proteinase step, most often incubation with a heat-stable protease in the presence of surfactants, to release the nucleic acid and degrade inhibitors that can interfere with downstream nucleic acid analysis. The amount of nucleic acid released is oftentimes minute because very little actual tissue is present in the slice and, in the case of an FFPE tissue slice, nucleic acids are frequently degraded. As a consequence, in the conventional methods, the nucleic acid most often needs to be concentrated before delivery to a downstream sensor in automated systems.
Non-toxic solutions for deparaffinization have been explored and improvements on nucleic acid recovery methods applicable on FFPE samples have been made available at the lab-scale level (e.g. WAXFREE™ Kit from Trimgen, ExpressArt FFPE Clear RNAready Kit from Amplification Technologies, BiOstic™ FFPE Tissue Isolation Kit from Mo Bio Laboratories, and QuickExtract™ FFPE DNA Extraction Kit from Epicentre).
One such improvement is described in WO2012/075133 and provides methods for in situ nucleic acid isolation from samples embedded in a hydrophobic matrix such as paraffin or a paraffin-blend. An emulsified lysate is hereto generated in the presence of a thermostable protease, and an additive selected from alkylene glycol, a poly-alkylene glycerol, or a block copolymer having an average molecular weight of 76 to 2900 Da, or a salt. Different additives are used for emulsifying the sample, including PEG200, PEG400, PEG1000, Brij30, Brij35P, Brij56 and Brij 76. The emulsified lysate is obtained in the presence of a mild chaotrope (e.g. urea or formamide) and heating. The method eliminates the need of physical separation of paraffin and the use of organic solvents such as xylene in a deparaffinization step. However, subsequent extraction of the nucleic acids from the emulsified lysate remains required for further downstream applications such as e.g. nucleic acid quantification by polymerase chain reaction, and such method might not be compatible with microfluidic systems
Integrating a nucleic acid extraction protocol into a microfluidic platform requires a great effort to optimize yield and minimize nucleic acid loss. Furthermore, extraction is also a time-consuming step in the sample preparation procedure. In addition, extraction introduces a size bias (loss of smaller fragments) in the eluted nucleic acids, which is especially problematic when isolating nucleic acids from FFPE samples, which contain degraded nucleic acids. Therefore, a method that is uniformly applicable for obtaining nucleic acids from a broad range of biological samples, including FFPE samples in a condition allowing automated microfluidic system processing and direct downstream analysis would provide a great advantage compared to existing methods. In particular, FFPE samples in a condition allowing automated microfluidic system processing and direct downstream analysis, without the risk of losing certain nucleic acid fragments and introducing a length and purity bias, would provide a great advantage compared to existing methods.
There is thus a need to improve the sample preparation process allowing automated high throughput processing and detection of nucleic acid in various biological samples.